CN117624178A - Aporphine alkaloid compound separated from glorious tree and application thereof - Google Patents
Aporphine alkaloid compound separated from glorious tree and application thereof Download PDFInfo
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- -1 Aporphine alkaloid compound Chemical class 0.000 title claims abstract description 18
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the field of biochemical medicines, and discloses an aporphine alkaloid compound separated from a dried-up tree and application thereof. The structure of the aporphine alkaloid compound is shown as a formula I:the compound is extracted and separated from the gloriosa, and in vitro cell experiments show that the compound has the effects of remarkably improving the decrease of the activity of MIN6 cells induced by palmitic acid and remarkably inhibiting the damage of islet cells induced by the palmitic acid. In addition, in vitro cell experiments also show that the composition has the effect of remarkably reducing normal hepatic cell triglyceride induced by palmitic acidThe content is increased, and the obvious lipid-lowering effect is confirmed.
Description
Technical Field
The invention belongs to the field of biochemical medicines, and particularly relates to an aporphine alkaloid compound separated from a gloriopsis cumingii tree and application thereof.
Background
Metabolic-related fatty liver disease (metabolic associated fatty liver disease, MAFLD) is a chronic, non-infectious disease based on liver fat accumulation combined with at least one of 3 conditions, overweight obesity, type 2 diabetes, metabolic dysfunction. Liver steatosis resulting from ectopic, excessive accumulation of fat in liver tissue is a benign onset of MAFLD, which stage has no apparent liver damage and can be reversed, but 10-20% of simple fatty liver can progress to NASH, further development can lead to cirrhosis and HCC, and may become the leading cause of liver transplantation in the future. The pathogenesis of MAFLD is extremely complex, and is not yet systematically elucidated. For a long time, the "second hit theory" proposed earlier has been widely accepted, wherein lipid accumulation in hepatocytes is the first hit and the key to the development of liver lipid deposition is insulin resistance (insulin resistance, IR). Steatosis caused by hepatocyte lipid accumulation increases the susceptibility of the liver to other damaging factors, namely secondary hits, leading to hepatocyte damage, inflammation and fibrosis. With the continuous and intensive research on MAFLD in recent years, it is found that the "secondary striking theory" is insufficient to explain the complex molecular mechanism and metabolic changes in MAFLD, and is gradually replaced by the "multiple striking theory". The "multiple hit theory" considers that a variety of causative factors such as gut dysregulation, trophic factors, inflammatory responses, genetic and epigenetic factors, IR and Oxidative Stress (OS), etc., act in parallel or sequentially in some synergistic fashion on genetically susceptible subjects to induce MAFLD. The proposal of the multiple striking theory makes people more fully aware of MAFLD, but the pathogenesis of MAFLD still needs to be studied intensively
The Litsea coreana (Lauraceae) is a evergreen arbor of the genus Litsea of the family Lauraceae, and is widely distributed in Asian tropical and subtropical regions, north America and south America subtropical regions. In China, it is mainly distributed in Guangdong, hainan, yunnan and so on. The leaves, roots and barks of the glorious tree are important medicinal plants in China, and have the effects of removing dampness and heat, diminishing inflammation, sterilizing, stopping bleeding, relieving pain and the like as traditional medicines.
Disclosure of Invention
In a first aspect, the present invention provides an aporphine alkaloid compound having a structure as shown in formula I:
the second aspect of the present invention provides a method for extracting the aporphine alkaloid compound, comprising the following steps:
(1) Drying and cutting the bark of the glehnia, heating and refluxing the bark of the glehnia with an organic solvent, filtering to obtain a total extract of the glehnia, and concentrating the total extract under reduced pressure until no alcohol smell exists to obtain the total extract; adding sulfuric acid solution into the obtained total extract to adjust the pH value to 2-3, adding ethyl acetate for extraction, removing an upper organic phase, recovering a lower layer, adding sodium hydroxide solution into the lower layer to adjust the pH value to 10-11, adding ethyl acetate for extraction, removing the lower layer, recovering an upper organic phase, and concentrating under reduced pressure to obtain ethyl acetate extract total extract, namely a total alkaloid part;
(2) Performing silica gel column chromatography on the obtained total extract, performing gradient elution by using dichloromethane-acetone with different proportions as eluent, collecting eluent eluted by dichloromethane-acetone=20:1 proportion, concentrating, and drying; drying the obtained fractions, purifying by Sephadex LH-20, wherein methanol is taken as an eluent, and each 50mL of methanol is taken as one fraction, so that 35 fractions are obtained, and the numbers of the 35 fractions are Fr.2b1-Fr.2b35 respectively;
(3) And combining the obtained fractions Fr.2b25-Fr.2b29, and further purifying by adopting semi-preparative high performance liquid chromatography to obtain the compound.
Further, the extractant used in step (1) is analytically pure methanol and/or 95% ethanol by volume; in the step (2), the particle size of silica gel used in the silica gel column chromatography is 200-300 meshes; in the step (2), the concentration is reduced pressure concentration, and the drying is vacuum freeze drying.
Further, the volume ratio of dichloromethane to acetone in the step (2) is sequentially 100:0, 20:1, 10:1, 4:1, 2:1 and 1:1.
Further, in the step (3), the high performance liquid chromatography column is se:Sup>A YMC-Pack ODS-A column, the volume ratio of the eluent is 22:78 of acetonitrile-containing 0.5% formic acid and 0.1% triethylamine water solution, the flow rate is 2 m/min, and the elution time is 40min; the retention time of this compound was 36.2min.
In a third aspect, the present invention provides an application of the aporphine alkaloid compound in preparation of a preparation for improving palmitic acid-induced decrease in MIN6 cell viability.
Further, in the application, the MIN6 cells are mouse islet beta cell lines.
In a fourth aspect, the present invention provides the use of an aporphine alkaloid compound as described above for the preparation of a formulation for reducing palmitic acid-induced elevation of normal hepatocyte triglyceride levels.
Further, in the application, the normal liver cells are human liver normal cells HL-7702 cells.
Further, the concentration of the aporphine alkaloid compound is 0.39-50 mu M.
The invention has the beneficial effects that:
the aporphine alkaloid compound is a compound with novel structure, which is separated from a gloriopsis cumingii tree. In vitro cell experiments show that the composition has the effects of remarkably improving the decrease of the activity of the palmitic acid-induced MIN6 cells and remarkably inhibiting the palmitic acid-induced islet cell injury. In addition, in vitro cell experiments also show that the composition has the effect of obviously reducing the triglyceride content of normal liver cells induced by palmitic acid, and has obvious lipid-lowering effect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is the effect of different treatments on palmitic acid induced MIN6 cell viability.
FIG. 2 is the effect of different treatments on palmitic acid induced HL-7702 cell triglyceride content.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Taking 20kg of the radix seu herba Litseae, drying, cutting into blocks, heating and reflux extracting with 95% ethanol, filtering to obtain radix Litseae total extract, concentrating under reduced pressure until no ethanol smell exists, and obtaining total extract; taking the obtained total extract, adding sulfuric acid solution to adjust the pH value to be 2-3, wherein the sulfuric acid solution can be 1% sulfuric acid solution; adding ethyl acetate for extraction, removing an upper organic phase, recovering a lower layer, adding sodium hydroxide solution into the lower layer to adjust the pH to be 10-11, adding ethyl acetate for extraction, removing the lower layer, recovering an upper organic phase, and concentrating under reduced pressure to obtain ethyl acetate extract total extract (68 g), namely a total alkaloid part;
taking the obtained total extract, loading the extract by using a silica gel dry method, namely firstly, extracting the total extract by using ethyl acetate, adding a small amount of methanol for dissolving, then adding a small amount of silica gel for uniformly stirring, grinding after the organic solvent volatilizes, performing column chromatography by using silica gel (200-300 meshes), performing gradient elution by using dichloromethane-acetone with different volume ratios as an eluent, eluting until the mixture is colorless, and collecting the mixture with the dichloromethane-acetone ratio of 20:1, concentrating under reduced pressure, and vacuum freeze drying to obtain dry powder effective component. The proportions of the eluents for each gradient and the nomenclature of the fractions obtained are shown in Table 1:
TABLE 1 nomenclature of fractions obtained for different gradient eluents
Purifying the obtained effective part (Fr.2) by Sephadex LH-20, wherein methanol is used as an eluent, and each 50mL of methanol is used as one fraction, and 35 fractions are obtained, and the numbers of the fractions are Fr.2b1-Fr.2b35 respectively; the resulting fractions fr.2b25-fr.2b29 were combined and further purified by semi-preparative high performance liquid chromatography to give 1 compound. The high performance liquid chromatography column is YMC-Pack ODS-A column, the eluent is acetonitrile-water solution (volume ratio is 22:78) containing 0.5% (V/V) formic acid and 0.1% (V/V) triethylamine, the flow rate is 2 m/min, and the elution time is 40min; the retention time of this compound was 36.2min.
The compound is identified as aporphine alkaloid, and the molecular formula is shown as formula I:
the high resolution mass spectrum data of the compounds of the present invention are shown in table 2.
TABLE 2 high resolution Mass Spectrometry data for the Compounds of the invention
The spectral data of the compounds of the present invention are shown in table 3.
TABLE 3 spectroscopic data for the compounds of the invention
1 H NMR test conditions were 600MHz, DMSO-d6; 13 c NMR test conditions were 150MHz, DMSO-d6.
The experimental method for improving palmitic acid-induced MIN6 cell viability by using the compound disclosed by the invention comprises the following steps:
(1) MIN6 cell resuscitation and culture: taking out in-80deg.C refrigerator, centrifuging to remove upper frozen stock solution, adding 10% foetal calf serum, 50 μm mercaptoethanol and 1% penicillin/streptomycin RPMI-1640 culture medium, and resuspending cells with relative humidity of 95%, 37deg.C, 5% CO 2 Is cultured in a incubator; according to the growth condition of the cells, changing fresh culture medium every 24-36 h, and carrying out passage or freezing storage when the cells grow to 80% -90%.
(2) The CCK-8 method detects the cell viability: uniformly inoculating MIN-6 cells with good growth state into 96-well plate according to 1×10 ratio 4 RPMI-1640 medium (10% FBS, 50. Mu.M mercaptoethanol, 10% P/S) was added to cells/mL at 100. Mu.L/well, and 96-well plates were placed in a 5% CO2 incubator at 37℃for 24 hours. The original medium was discarded and divided into a blank (CK) model control group (300 μm PA added) and an experimental group given 300 μm PA, while the compound to be screened was given an intervention treatment. According to 1X 10 4 RPMI-1640 medium is added into cells/mL, 100 mu L/hole of the RPMI-1640 medium is added into the culture medium, the culture is carried out for 24 hours, the concentration gradient of each group is 5 parallel compound holes, and the average value of a blank group is used as a control.
(4) After MIN-6 cells were subjected to the above conditional intervention, CCK-8 solution was added to 96 wells at 100. Mu.L per well. Then, the mixture was placed in an incubator at 37℃with 5% CO2, incubated for 1 hour, and finally the Optical Density (OD) was measured at a wavelength of 450nm using an enzyme-labeled instrument. And cell viability was calculated.
The experimental results are shown in Table 4 and FIG. 1.
TABLE 4 Effect of different treatments on MIN-6 cell viability
Note that: comparison of blank control with model control # P<0.05， ## P<0.01， ### P<0.001；
Experimental group was compared with model control group with P <0.05, P <0.01, P <0.001.
The aporphine alkaloid compound separated from the effective part obtained by the invention is diluted to 0.39 mu M from 50 mu M to 2 times in sequence, and the activity of the palmitic acid-induced MIN6 cells is influenced under 8 treatment concentrations. As shown in fig. 1: CK is a blank (no compound and palmitic acid treatment) and PA is a model control (PA concentration 300 μm/ml). Therefore, the blank control group has significant difference compared with the model control group, which indicates that the model group has successful palmitic acid administration modeling; compared with the model control group, the compound remarkably improves the reduction of cell viability caused by palmitic acid in the concentration range of 0.39-6.25 mu M, wherein the effect of the compound concentration of 1.56 mu M is optimal.
The experimental method for reducing the triglyceride content of normal hepatocytes induced by palmitic acid by the compound comprises the following steps:
(1) Resuscitating and culturing HL-7702 cells: taking out in a refrigerator at-80deg.C, centrifuging to remove upper frozen stock solution, adding RPMI1640 culture medium containing 10% foetal calf serum and 1% double antibody to re-suspend cells, and culturing in incubator with relative humidity of 95%, 37deg.C and 5% CO 2; changing fresh culture medium every 24-36 h according to the growth condition of cells, and after the cells grow to 80% -90%, carrying out passage or freezing storage, and after stable passage for 2 times, using the cells suspension according to the ratio of 2.0X10 5 cells/ml density was inoculated uniformly into 12-well plates at 1ml per well and cultured in cell culture chambers.
(2) The wells were divided into the following groups: blank (10% BSA), model (300. Mu.M PA-BSA), dosing (300. Mu.M PA-BSA+0.3125/0.625/1.25/2.5/5/10. Mu.M aporphine alkaloid), 3 multiplex wells per group. After the inoculated 12-well plate is continuously cultured for 24 hours, the original culture solution is removed, and BSA, PA-BSA and aporphine alkaloid with target concentration are mixed, and the culture treatment is carried out for 24 hours. The cell culture broth was discarded, washed 2 times with ice-chilled PBS, and the cells were collected.
(3) Intracellular TG levels were measured: the intracellular TG levels of each treatment group were determined using a tissue cell Triglyceride (TG) enzyme assay kit (beijing plley gene technologies, inc., E1013).
The experimental results are shown in Table 5 and FIG. 2.
TABLE 5 Effect of different treatments on palmitic acid induced HL-7702 cell triglyceride content
| Compound treatment method | Triglyceride content (μg/mg) |
| Blank control group (CK) | 17.95 |
| Model group | 84.76 ### |
| Experiment group 1 (PA+ Compound-0.3125. Mu.M) | 61.81 ** |
| Experiment group 2 (PA+ Compound-0.625. Mu.M) | 79.53 * |
| Experiment group 3 (PA+ Compound-1.25. Mu.M) | 91.79 |
| Experiment group 4 (PA+ Compound-2.5. Mu.M) | 92.44 |
| Experiment group 5 (PA+ Compound-5. Mu.M) | 95.15 |
| Experiment group 6 (PA+ Compound-10. Mu.M) | 106.34 |
Note that: comparison of blank control group and model group # P<0.05， ## P<0.01， ### P<0.001；
Experimental group and model group were compared with P <0.05, P <0.01, P <0.001.
The aporphine alkaloid compound separated from the effective part obtained by the invention is diluted to 0.3125 mu M from 10 mu M to 2 times in sequence, and the effect on the triglyceride content of the HL-7702 cells induced by palmitic acid is achieved under 6 treatment concentrations. As shown in fig. 2: the blank group (without treatment by PA and compound) has significant difference compared with the model group (PA concentration is 300 mu M), which indicates that the model group has successful palmitic acid administration modeling; compared with a model group, the compound obviously reduces the triglyceride content of normal hepatic cells induced by palmitic acid at the concentration of 0.3125 mu M and 0.625 mu M, which proves that the compound has obvious lipid-lowering activity.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. An aporphine alkaloid compound is characterized in that the structure is shown as a formula I:
2. the extraction method of the aporphine alkaloid compound according to claim 1, comprising the steps of:
(1) Drying and cutting the bark of the glehnia, heating and refluxing the bark of the glehnia with an organic solvent, filtering to obtain a total extract of the glehnia, and concentrating the total extract under reduced pressure until no alcohol smell exists to obtain the total extract; adding sulfuric acid solution into the obtained total extract to adjust the pH value to 2-3, adding ethyl acetate for extraction, removing an upper organic phase, recovering a lower layer, adding sodium hydroxide solution into the lower layer to adjust the pH value to 10-11, adding ethyl acetate for extraction, removing the lower layer, recovering an upper organic phase, and concentrating under reduced pressure to obtain ethyl acetate extract total extract, namely a total alkaloid part;
(2) Performing silica gel column chromatography on the obtained total extract, performing gradient elution by using dichloromethane-acetone with different proportions as eluent, collecting eluent eluted by dichloromethane-acetone=20:1 proportion, concentrating, and drying; drying the obtained fractions, purifying by Sephadex LH-20, wherein methanol is taken as an eluent, and each 50mL of methanol is taken as one fraction, so that 35 fractions are obtained, and the numbers of the 35 fractions are Fr.2b1-Fr.2b35 respectively;
(3) And combining the obtained fractions Fr.2b25-Fr.2b29, and further purifying by adopting semi-preparative high performance liquid chromatography to obtain the compound.
3. The extraction method according to claim 2, wherein the extractant used in step (1) is analytically pure methanol and/or 95% ethanol by volume; in the step (2), the particle size of silica gel used in the silica gel column chromatography is 200-300 meshes; in the step (2), the concentration is reduced pressure concentration, and the drying is vacuum freeze drying.
4. The extraction method according to claim 2, wherein the dichloromethane-acetone volume ratio in step (2) is 100:0, 20:1, 10:1, 4:1, 2:1, 1:1 in order.
5. The method according to claim 2, wherein the high performance liquid chromatography column in the step (3) is se:Sup>A YMC-Pack ODS-se:Sup>A column, the volume ratio of eluent is 22:78 acetonitrile-containing 0.5% formic acid and 0.1% triethylamine aqueous solution, the flow rate is 2mL/min, and the elution time is 40min; the retention time of this compound was 36.2min.
6. Use of an aporphine alkaloid compound of claim 1 for the preparation of a formulation for ameliorating palmitic acid-induced decrease in MIN6 cell viability.
7. The use of claim 6, wherein in said use, said MIN6 cells are a mouse islet beta cell line.
8. Use of an aporphine alkaloid compound of claim 1 for the preparation of a formulation for reducing palmitic acid-induced elevation of normal hepatocellular triglyceride levels.
9. The use according to claim 8, wherein in said use, the normal liver cells are human liver normal cells HL-7702 cells.
10. The use according to claim 6 or 9, characterized in that the concentration of aunt on aporphine alkaloid compound is 0.39-50 μm.
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